Seed development, unique to higher plants, involves embryo development as well as physiological adaptation processes that occur within the seed to ensure the survival of the developing seedling upon germination. After fertilization, there is rapid growth and differentiation of the embryo and endosperm, after which nutritive reserves accumulate during the maturation stage of seed development. These reserves are stored during a period of developmental arrest for later use by the developing seedling. This period of arrest occurs prior to the desiccation phase of seed development.
Several classes of seed proteins, including storage proteins, lectins, and trypsin inhibitors, accumulate during embryogenesis. The main function of seed storage proteins is to accumulate during embryogenesis and to store carbon and nitrogen reserves for the developing seedling upon germination. These proteins, as well as many of the genes encoding them, have been studied extensively (for review see Shotwell et al. (1989) in The Biochemistry of Plants, 15, Academic Press, N.Y., 297).
Genes encoding seed storage proteins are highly regulated and differentially expressed during seed development. Expression is temporally regulated with mRNA accumulating rapidly during the maturation phase of embryogenesis. This expression is also tissue-specific, occurring primarily in the cotyledons or endosperm of the developing seeds. The resulting storage proteins are processed and targeted to protein bodies, in which the storage proteins remain during desiccation and dormancy of the embryo. Upon germination, the seedling uses these storage proteins as a source of carbon and nitrogen (Higgins (1984) Ann. Rev. Plant Physiol. 35, 191).
Seed proteins, including storage proteins, lectins and trypsin inhibitors, are encoded by nonhomologous multigene families that are not amplified or structurally altered during development (for review see Goldberg et al. (1989) Cell 56, 149). These genes are temporally and spatially regulated but not necessarily linked. Although post-transcriptional mechanisms act to control the accumulation of some of these proteins, regulation occurs primarily at the transcriptional level. Accordingly, seed protein genes provide an excellent system to provide genetic regulatory elements, especially those elements which confer tissue specificity, temporal regulation, and responsiveness to environmental and chemical cues.
Observations of temporal and spatial regulation of seed protein genes has suggested that seed protein genes are regulated in part by common cellular factors known as trans-acting factors. However, since quantitative and qualitative differences exist in the expression patterns of individual seed protein genes, more specific factors must also exist to provide a means for differential expression patterns between these groups of seed proteins. Patterns of differential expression have been observed between the rapeseed major seed storage proteins, cruciferin and napin (Crouch et al. (1981) Planta 153, 64; Finkelstein et al. (1985) Plant Physiol. 78, 630), and among individual members of the soybean Kunitz trypsin inhibitor gene family (Jofuku et al. (1989) Plant Cell 1, 1079). A comparison of the soybean major seed storage protein genes showed a difference in timing and cell-type specificity of the expression of .beta.-conglycinin (7S) and glycinin (11S). The 7S subunit mRNA appeared several days before the 11S mRNA. Furthermore, while members of the glycinin gene family were all activated simultaneously (Nielsen et al. (1989) Plant Cell 1, 313), members of the .beta.-conglycinin gene family were differentially regulated (Barker et al. (1988) Proc. Natl. Acad. Sci. USA 85, 458; Chen et al. (1989) Dev. Genet. 10, 112). Each of these genes contain a different array of cis-regulatory elements which confer differential expression patterns between, and within, these gene families.
Helianthinin is the major 11S globulin seed storage protein of sunflower (Helianthus annuus). Helianthinin expression, like that of other seed storage proteins, is tissue-specific and under developmental control. However, the helianthin regulatory elements which confer such specificity have heretofore never been identified. Helianthinin mRNA is first detected in embryos 7 days post flowering (DPF) with maximum levels of mRNA reached at 12-15 DPF, after which the level of helianthinin transcripts begins to decline. In mature seeds or in germinating seedlings helianthinin transcripts are absent. Helianthinin polypeptide accumulation is rapid from 7 DPF through 19 DPF but slows as the seed reaches later maturation stages (Allen et al. (1985) Plant Mol. Biol. 5, 165).
Helianthinin, like most seed proteins, is encoded by a small gene family. At least two divergent subfamilies are known, and are designated Ha2 and Ha10. Two clones, HaG3-A and HaG3-D, representing non-allelic members of the Ha2 subfamily, have been isolated and partially characterized (Vonder Haar et al. (1988) Gene 74, 433). However, a detailed analysis of the regulatory elements of these or any other helianthinin genes had not been known until now.
It has been found in accordance with the present invention that regulatory elements from helianthinin genes can direct seed-specific gene expression, root-specific gene expression, abscisic acid-responsive gene expression, and/or temporally-altered gene expression. These regulatory elements enable the controlled expression of specific gene products in transgenic plants. The present invention provides greater control of gene expression in transgenic plants, thus allowing improved seed quality, improved tolerance to environmental conditions such as drought, and better control of herbicide resistance genes.